This proposal extends the structural studies of acyl-CoA dehydrogenases electron transfer flavoprotein (ETF), and ETF-ubiquinone oxidoreductase (ETF-QO). ADs are an enzyme family involved in the first step in mitochondrial fatty acid (-oxidation and in the catabolism of some amino acids. ETF is the physiological oxidant of ADs and sarcosine dehydrogenase. Sarcosine dehydrogenase is not a member of the AD family and functions in choline metabolism and transfers a 1-carbon unit to tetrahydrofolate. ETF funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF- QO, a membrane-bound, iron-sulfur flavoprotein. Fatty acid (-oxidation is the major energy-producing process in the liver, heart, and muscle. The rate of this process can be altered by diet (starvation), physiological status (pregnancy), or diseases (diabetes). The critical metabolic roles of ADs, ETF, ETF-QO, and sarcosine dehydrogenase are illustrated by the severity of human diseases resulting from inherited deficiencies of these enzymes. Crystal structures have been determined for short- and medium-chain acyl-CoA dehydrogenases (MCAD), isovaleryl-CoA dehydrogenase, and glutaryl-CoA dehydrogenase (GCD). It is proposed: 1) to continue the studies of mutants of MCAD and its complexes with substrate analog/inhibitors and to determine the structure of human long chain acyl-CoA dehydrogenase in order to confirm the catalytic base and determine the structural basis for long-chain specificity; 2) to continue the structural studies of GCD in order to study the mechanism of the decarboxylation reaction; and 3) to complete the structure determination of isobutyryl-CoA dehydrogenase (involved in valine metabolism) and short/branched chain acyl-CoA dehydrogenase (involved in isoleucine catabolism) to determine their branched chain-substrate specificities. Structure of porcine ETF-QO has been determined with and without a bound ubiquinone. It is also proposed: 4) to extend studies of ETF-QO to its complexes with ubiquinone analogs and mutants, in order to determine the role of the 4Fe-4S cluster and 5) to initiate the structure determination of sarcosine dehydrogenase in order to determine the mechanism of oxidative demethylation and 1-carbon transfer reactions. It is further proposed 6) to extend the structural studies of E. coli dienoyl-CoA reductase, an auxiliary enzyme for unsaturated fatty acid metabolism, to its mutants and complexes with different stereoisomeric substrates to confirm the roles of putative catalytic residues and to determine the structural basis for metabolism of different stereoisomers.